Beta-defensin index: A functional biomarker for oral cancer detection.

There is an unmet clinical need for a non-invasive and cost-effective test for oral squamous cell carcinoma (OSCC) that informs clinicians when a biopsy is warranted. Human beta-defensin 3 (hBD-3), an epithelial cell-derived anti-microbial peptide, is pro-tumorigenic and overexpressed in early-stage OSCC compared to hBD-2. We validate this expression dichotomy in carcinoma in situ and OSCC lesions using immunofluorescence microscopy and flow cytometry. The proportion of hBD-3/hBD-2 levels in non-invasively collected lesional cells compared to contralateral normal cells, obtained by ELISA, generates the beta-defensin index (BDI). Proof-of-principle and blinded discovery studies demonstrate that BDI discriminates OSCC from benign lesions. A multi-center validation study shows sensitivity and specificity values of 98.2% (95% confidence interval [CI] 90.3-99.9) and 82.6% (95% CI 68.6-92.2), respectively. A proof-of-principle study shows that BDI is adaptable to a point-of-care assay using microfluidics. We propose that BDI may fulfill a major unmet need in low-socioeconomic countries where pathology services are lacking.

Distinct SARS-CoV-2 specific NLRP3 and IL-1ß responses in T cells of aging patients during acute COVID-19 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19) that presents with varied clinical manifestations ranging from asymptomatic or mild infections and pneumonia to severe cases associated with cytokine storm, acute respiratory distress syndrome (ARDS), and even death. The underlying mechanisms contributing to these differences are unclear, although exacerbated inflammatory sequelae resulting from infection have been implicated. While advanced aging is a known risk factor, the precise immune parameters that determine the outcome of SARS-CoV-2 infection in elderly individuals are not understood. Here, we found aging-associated (age ≥61) intrinsic changes in T cell responses when compared to those from individuals aged ≤ 60, even among COVID-positive patients with mild symptoms. Specifically, when stimulated with SARS-CoV-2 peptides in vitro, peripheral blood mononuclear cell (PBMC) CD4+ and CD8+ T cells from individuals aged ≥61 showed a diminished capacity to produce IFN-γ and IL-1ß. Although they did not have severe disease, aged individuals also showed a higher frequency of PD-1+ cells and significantly diminished IFN-γ/PD-1 ratios among T lymphocytes upon SARS-CoV-2 peptide stimulation. Impaired T cell IL-1ß expression coincided with reduced NLRP3 levels in T lymphocytes. However, the expression of these molecules was not affected in the monocytes of individuals aged ≥61. Together, these data reveal SARS-CoV-2-specific CD4+ and CD8+ T-cell intrinsic cytokine alterations in the individuals older than 61 and may provide new insights into dysregulated COVID-directed immune responses in the elderly.

Inflammation, Immune Senescence, and Dysregulated Immune Regulation in the Elderly.

An optimal immune response requires the appropriate interaction between the innate and the adaptive arms of the immune system as well as a proper balance of activation and regulation. After decades of life, the aging immune system is continuously exposed to immune stressors and inflammatory assaults that lead to immune senescence. In this review, we will discuss inflammaging in the elderly, specifically concentrating on IL-6 and IL-1b in the context of T lymphocytes, and how inflammation is related to mortality and morbidities, specifically cardiovascular disease and cancer. Although a number of studies suggests that the anti-inflammatory cytokine TGF-b is elevated in the elderly, heightened inflammation persists. Thus, the regulation of the immune response and the ability to return the immune system to homeostasis is also important. Therefore, we will discuss cellular alterations in aging, concentrating on senescent T cells and CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) in aging.

Fungal Colonization and Infections-Interactions with Other Human Diseases.

Candida albicans is a commensal fungus that asymptomatically colonizes the skin and mucosa of 60% of healthy individuals. Breaches in the cutaneous and mucosal barriers trigger candidiasis that ranges from asymptomatic candidemia and mucosal infections to fulminant sepsis with 70% mortality rates. Fungi influence at least several diseases, in part by mechanisms such as the production of pro-carcinogenic agents, molecular mimicking, and triggering of the inflammation cascade. These processes impact the interactions among human pathogenic and resident fungi, the bacteriome in various organs/tissues, and the host immune system, dictating the outcomes of invasive infections, metabolic diseases, and cancer. Although mechanistic investigations are at stages of infancy, recent studies have advanced our understanding of host-fungal interactions, their role in immune homeostasis, and their associated pathologies. This review summarizes the role of C. albicans and other opportunistic fungi, specifically their association with various diseases, providing a glimpse at the recent developments and our current knowledge in the context of inflammatory-bowel disease (IBD), cancers, and COVID-19. Two of the most common human diseases where fungal interactions have been previously well-studied are cancer and IBD. Here we also discuss the emerging role of fungi in the ongoing and evolving pandemic of COVID-19, as it is relevant to current health affairs.

Regulation of IL-17A-Producing Cells in Skin Inflammatory Disorders.

This review focuses on the IL-17A family of cytokines produced by T lymphocytes and other immune cells and how they are involved in cutaneous pathogenic responses. It will also discuss cutaneous dysbiosis and FOXP3+ regulatory T cells in the context of inflammatory conditions linked to IL-17 responses in the skin. Specifically, it will review key literature on chronic mucocutaneous candidiasis and psoriasis.

The Role of Dectin-1 Signaling in Altering Tumor Immune Microenvironment in the Context of Aging.

An increased accumulation of immune-dysfunction-associated CD4+Foxp3+ regulatory T cells (Tregs) is observed in aging oral mucosa during infection. Here we studied the function of Tregs during oral cancer development in aging mucosa. First, we found heightened proportions of Tregs and myeloid-derived suppressor cells (MDSC) accumulating in mouse and human oral squamous cell carcinoma (OSCC) tissues. Using the mouse 4-Nitroquinoline 1-oxide(4-NQO) oral carcinogenesis model, we found that tongues of aged mice displayed increased propensity for epithelial cell dysplasia, hyperplasia, and accelerated OSCC development, which coincided with significantly increased abundance of IL-1ß, Tregs, and MDSC in tongues. Partial depletion of Tregs reduced tumor burden. Moreover, fungal abundance and dectin-1 signaling were elevated in aged mice suggesting a potential role for dectin-1 in modulating immune environment and tumor development. Confirming this tenet, dectin-1 deficient mice showed diminished IL-1ß, reduced infiltration of Tregs and MDSC in the tongues, as well as slower progression and reduced severity of tumor burden. Taken together, these data identify an important role of dectin-1 signaling in establishing the intra-tumoral immunosuppressive milieu and promoting OSCC tumorigenesis in the context of aging.

Naïve CD4+ T Cell Lymphopenia and Apoptosis in Chronic Hepatitis C Virus Infection Is Driven by the CD31+ Subset and Is Partially Normalized in Direct-Acting Antiviral Treated Persons.

Background: The mechanisms underlying naïve CD4+ lymphopenia during chronic Hepatitis C Virus (HCV) infection are unclear. Whether direct-acting antiviral (DAA) therapy restores peripheral naïve CD4+ T cell numbers and function is unknown. Methods: We enumerated frequencies and counts of peripheral naïve CD4+, CD4+CD31+ and CD4+CD31- T cells by flow cytometry in a cross sectional analysis comparing chronic HCV infected (n=34), DAA-treated(n=29), and age-range matched controls (n=25), as well as in a longitudinal cohort of HCV DAA treated persons (n=16). The cross-sectional cohort was stratified by cirrhosis state. Cell apoptosis/survival (AnnexinV+7AAD+/BCL-2 labeling) and cell cycle entry (Ki67 expression) of CD31+ and CD31- naïve CD4+ T cells was analyzed directly ex vivo and following 3 and 5 days of in vitro culture with media, interleukin (IL) -7 or CD3/CD28 activator. Results: In the cross-sectional cohort, naïve CD4+ proportions were lower in chronic HCV infected persons compared to controls and DAA-treated persons, an effect in part attributed to cirrhosis. Age was associated with naïve cell counts and proportions in HCV infected and treated persons as well. Naïve CD4+ cell proportions negatively correlated with plasma levels of soluble CD14 following therapy in DAA-treated persons. Naïve CD4+ cells from HCV infected persons exhibited greater direct ex vivo apoptosis and cell-cycling compared to cells from DAA-treated persons and controls, and this was localized to the CD4+CD31+ subset. On the other hand, no remarkable differences in expression of BCL-2 or IL-7 Receptor (CD127) at baseline or following in vitro media or IL7 containing culture were observed. In the longitudinal cohort, naïve CD4+CD31+/CD31- ratio tended to increase 24 weeks after DAA therapy initiation. Conclusions: Activation and apoptosis of peripheral naïve CD4+CD31+ T cells appear to contribute to naïve CD4+ lymphopenia in chronic HCV infection, and this defect is partially reversible with HCV DAA therapy. Age and cirrhosis -associated naïve CD4+ lymphopenia is present both before and after HCV DAA therapy. These findings have implications for restoration of host immune function after DAA therapy.

IL-1ß-MyD88-mTOR Axis Promotes Immune-Protective IL-17A+Foxp3+ Cells During Mucosal Infection and Is Dysregulated With Aging.

CD4+Foxp3+Tregs maintain immune homeostasis, but distinct mechanisms underlying their functional heterogeneity during infections are driven by specific cytokine milieu. Here we show that MyD88 deletion in Foxp3+ cells altered their function and resulted in increased fungal burden and immunopathology during oral Candida albicans (CA) challenge. Excessive inflammation due to the absence of MyD88 in Tregs coincided with a reduction of the unique population of IL-17A expressing Foxp3+ cells (Treg17) and an increase in dysfunctional IFN-γ+/Foxp3+ cells (TregIFN-γ) in infected mice. Failure of MyD88-/- Tregs to regulate effector CD4+ T cell functions correlated with heightened levels of IFN-γ in CD4+ T cells, as well as increased infiltration of inflammatory monocytes and neutrophils in oral mucosa in vivo. Mechanistically, IL-1ß/MyD88 signaling was required for the activation of IRAK-4, Akt, and mTOR, which led to the induction and proliferation of Treg17 cells. In the absence of IL-1 receptor signaling, Treg17 cells were reduced, but IL-6-driven expansion of TregIFN-γ cells was increased. This mechanism was physiologically relevant during Candida infection in aged mice, as they exhibited IL-1 receptor/MyD88 defect in Foxp3+ cells, loss of p-mTORhighTreg17 cells and reduced levels of IL-1ß in oral mucosa, which coincided with persistent tongue inflammation. Concurrent with Treg dysfunction, aging was associated with increased CD4+ T cell hyperactivation and heightened levels of IL-6 in mice and humans in oral mucosa in vivo. Taken together, our data identify IL-1ß/MyD88/Treg axis as a new component that modulates inflammatory responses in oral mucosa. Also, dysregulation of this axis in an aging immune system may skew host defense towards an immunopathological response in mucosal compartments.

Innate immune mechanisms to oral pathogens in oral mucosa of HIV-infected individuals.

A crucial aspect of mucosal HIV transmission is the interaction between HIV, the local environmental milieu and immune cells. The oral mucosa comprises many host cell types including epithelial cells, CD4 + T cells, dendritic cells and monocytes/macrophages, as well as a diverse microbiome predominantly comprising bacterial species. While the oral epithelium is one of the first sites exposed to HIV through oral-genital contact and nursing infants, it is largely thought to be resistant to HIV transmission via mechanisms that are still unclear. HIV-1 infection is also associated with predisposition to secondary infections, such as tuberculosis, and other diseases including cancer. This review addresses the following questions that were discussed at the 8th World Workshop on Oral Health and Disease in AIDS held in Bali, Indonesia, 13 September -15 September 2019: (a) How does HIV infection affect epithelial cell signalling? (b) How does HIV infection affect the production of cytokines and other innate antimicrobial factors, (c) How is the mucosal distribution and function of immune cells altered in HIV infection? (d) How do T cells affect HIV (oral) pathogenesis and cancer? (e) How does HIV infection lead to susceptibility to TB infections?

Microbiome Dependent Regulation of Tregs and Th17 Cells in Mucosa.

Mammals co-exist with resident microbial ecosystem that is composed of an incredible number and diversity of bacteria, viruses and fungi. Owing to direct contact between resident microbes and mucosal surfaces, both parties are in continuous and complex interactions resulting in important functional consequences. These interactions govern immune homeostasis, host response to infection, vaccination and cancer, as well as predisposition to metabolic, inflammatory and neurological disorders. Here, we discuss recent studies on direct and indirect effects of resident microbiota on regulatory T cells (Tregs) and Th17 cells at the cellular and molecular level. We review mechanisms by which commensal microbes influence mucosa in the context of bioactive molecules derived from resident bacteria, immune senescence, chronic inflammation and cancer. Lastly, we discuss potential therapeutic applications of microbiota alterations and microbial derivatives, for improving resilience of mucosal immunity and combating immunopathology.

Cycling CD4+ T cells in HIV-infected immune nonresponders have mitochondrial dysfunction.

Immune nonresponder (INR) HIV-1-infected subjects are characterized by their inability to reconstitute the CD4+ T cell pool after antiretroviral therapy. This is linked to poor clinical outcome. Mechanisms underlying immune reconstitution failure are poorly understood, although, counterintuitively, INRs often have increased frequencies of circulating CD4+ T cells in the cell cycle. While cycling CD4+ T cells from healthy controls and HIV+ patients with restored CD4+ T cell numbers complete cell division in vitro, cycling CD4+ T cells from INRs do not. Here, we show that cells with the phenotype and transcriptional profile of Tregs were enriched among cycling cells in health and in HIV infection. Yet there were diminished frequencies and numbers of Tregs among cycling CD4+ T cells in INRs, and cycling CD4+ T cells from INR subjects displayed transcriptional profiles associated with the impaired development and maintenance of functional Tregs. Flow cytometric assessment of TGF-ß activity confirmed the dysfunction of Tregs in INR subjects. Transcriptional profiling and flow cytometry revealed diminished mitochondrial fitness in Tregs among INRs, and cycling Tregs from INRs had low expression of the mitochondrial biogenesis regulators peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1α) and transcription factor A for mitochondria (TFAM). In vitro exposure to IL-15 allowed cells to complete division, restored the expression of PGC1α and TFAM, and regenerated mitochondrial fitness in the cycling Tregs of INRs. Our data suggest that rescuing mitochondrial function could correct the immune dysfunction characteristic of Tregs in HIV-1-infected subjects who fail to restore CD4+ T cells during antiretroviral therapy.

Role of Short Chain Fatty Acids in Controlling Tregs and Immunopathology During Mucosal Infection.

Interactions between mucosal tissues and commensal microbes control appropriate host immune responses and inflammation, but very little is known about these interactions. Here we show that the depletion of resident bacteria using antibiotics (Abx) causes oral and gut immunopathology during oropharyngeal candidiasis (OPC) infection. Antibiotic treatment causes reduction in the frequency of Foxp3+ regulatory cells (Tregs) and IL-17A producers, with a concomitant increase in oral tissue pathology. While C. albicans (CA) is usually controlled in the oral cavity, antibiotic treatment led to CA dependent oral and gut inflammation. A combination of short chain fatty acids (SCFA) controlled the pathology in Abx treated mice, correlating to an increase in the frequency of Foxp3+, IL-17A+, and Foxp3+IL-17A+ double positive (Treg17) cells in tongue and oral draining lymph nodes. However, SCFA treatment did not fully reverse the gut inflammation suggesting that resident microbiota have SCFA independent homeostatic mechanisms in gut mucosa. We also found that SCFA potently induce Foxp3 and IL-17A expression in CD4+ T cells, depending on the cytokine milieu in vitro. Depletion of Tregs alone in FDTR mice recapitulated oral inflammation in CA infected mice, showing that Abx mediated reduction of Tregs was involved in infection induced pathology. SCFA did not control inflammation in Treg depleted mice in CA infected FDTR mice, showing that Foxp3+ T cell induction was required for the protective effect mediated by SCFA. Taken together, our data reveal that SCFA derived from resident bacteria play a critical role in controlling immunopathology by regulating T cell cytokines during mucosal infections. This study has broader implications on protective effects of resident microbiota in regulating pathological infections.

Identification of Casz1 as a Regulatory Protein Controlling T Helper Cell Differentiation, Inflammation, and Immunity.

While T helper (Th) cells play a crucial role in host defense, an imbalance in Th effector subsets due to dysregulation in their differentiation and expansion contribute to inflammatory disorders. Here, we show that Casz1, whose function is previously unknown in CD4+ T cells, coordinates Th differentiation in vitro and in vivo. Casz1 deficiency in CD4+ T cells lowers susceptibility to experimental autoimmune encephalomyelitis, consistent with the reduced frequency of Th17 cells, despite an increase in Th1 cells in mice. Loss of Casz1 in the context of mucosal Candida infection severely impairs Th17 and Treg responses, and lowers the ability of the mice to clear the secondary infection. Importantly, in both the models, absence of Casz1 causes a significant diminution in IFN-γ+IL-17A+ double-positive inflammatory Th17 cells (Th1* cells) in tissues in vivo. Transcriptome analyses of CD4+ T cells lacking Casz1 show a signature consistent with defective Th17 differentiation. With regards to Th17 differentiation, Casz1 limits repressive histone marks and enables acquisition of permissive histone marks at Rorc, Il17a, Ahr, and Runx1 loci. Taken together, these data identify Casz1 as a new Th plasticity regulator having important clinical implications for autoimmune inflammation and mucosal immunity.

Kaposi's sarcoma-associated herpesvirus infection promotes differentiation and polarization of monocytes into tumor-associated macrophages.

Tumor associated macrophages (TAMs) promote angiogenesis, tumor invasion and metastasis, and suppression of anti-tumor immunity. These myeloid cells originate from monocytes, which differentiate into TAMs upon exposure to the local tumor microenvironment. We previously reported that Kaposi's sarcoma-associated herpes virus (KSHV) infection of endothelial cells induces the cytokine angiopoietin-2 (Ang-2) to promote migration of monocytes into tumors. Here we report that KSHV infection of endothelial cells induces additional cytokines including interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-13 (IL-13) that drive monocytes to differentiate and polarize into TAMs. The KSHV-induced TAMs not only express TAM-specific markers such as CD-163 and legumain (LGMN) but also display a gene expression profile with characteristic features of viral infection. More importantly, KSHV-induced TAMs enhance tumor growth in nude mice. These results are consistent with the strong presence of TAMs in Kaposi's sarcoma (KS) tumors. Therefore, KSHV infection of endothelial cells generates a local microenvironment that not only promotes the recruitment of monocytes but also induces their differentiation and polarization into TAMs. These findings reveal a new mechanism of KSHV contribution to KS tumor development.

Mucosal Regulatory T Cells and T Helper 17 Cells in HIV-Associated Immune Activation.

Residual mucosal inflammation along with chronic systemic immune activation is an important feature in individuals infected with human immunodeficiency virus (HIV), and has been linked to a wide range of co-morbidities, including malignancy, opportunistic infections, immunopathology, and cardiovascular complications. Although combined antiretroviral therapy (cART) can reduce plasma viral loads to undetectable levels, reservoirs of virus persist, and increased mortality is associated with immune dysbiosis in mucosal lymphoid tissues. Immune-based therapies are pursued with the goal of improving CD4(+) T-cell restoration, as well as reducing chronic immune activation in cART-treated patients. However, the majority of research on immune activation has been derived from analysis of circulating T cells. How immune cell alterations in mucosal tissues contribute to HIV immune dysregulation and the associated risk of non-infectious chronic complications is less studied. Given the significant differences between mucosal T cells and circulating T cells, and the immediate interactions of mucosal T cells with the microbiome, more attention should be devoted to mucosal immune cells and their contribution to systemic immune activation in HIV-infected individuals. Here, we will focus on mucosal immune cells with a specific emphasis on CD4(+) T lymphocytes, such as T helper 17 cells and CD4(+)Foxp3(+) regulatory T cells (Tregs), which play crucial roles in maintaining mucosal barrier integrity and preventing inflammation, respectively. We hypothesize that pro-inflammatory milieu in cART-treated patients with immune activation significantly contributes to enhanced loss of Th17 cells and increased frequency of dysregulated Tregs in the mucosa, which in turn may exacerbate immune dysfunction in HIV-infected patients. We also present initial evidence to support this hypothesis. A better comprehension of how pro-inflammatory milieu impacts these two types of cells in the mucosa will shed light on mucosal immune dysfunction and HIV reservoirs, and lead to novel ways to restore immune functions in HIV(+) patients.

FAD-I, a Fusobacterium nucleatum Cell Wall-Associated Diacylated Lipoprotein That Mediates Human Beta Defensin 2 Induction through Toll-Like Receptor-1/2 (TLR-1/2) and TLR-2/6.

We previously identified a cell wall-associated protein from Fusobacterium nucleatum, a Gram-negative bacterium of the oral cavity, that induces human beta defensin 2 (hBD-2) in primary human oral epithelial cells (HOECs) and designated it FAD-I (Fusobacterium-associated defensin inducer). Here, we report differential induction of hBD-2 by different strains of F. nucleatum; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-borne fadI from the highly hBD-2-inducing strains in a ΔfadI mutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principal F. nucleatum agent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blocked F. nucleatum induction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed in Escherichia coli triggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated from F. nucleatum ATCC 25586. Tandem mass spectrometry revealed a diacylglycerol modification at the cysteine residue in position 16 for both nFAD-I and rFAD-I. Cysteine-to-alanine substitution abrogated FAD-I's ability to induce hBD-2. Finally, FAD-I activation of hBD-2 expression was mediated via both Toll-like receptor-1/2 (TLR-1/2) and TLR-2/6 heterodimerization. Microbial molecules like FAD-I may be utilized in novel therapeutic ways to bolster the host innate immune response at mucosal surfaces.

Origin and functions of pro-inflammatory cytokine producing Foxp3+ regulatory T cells.

CD4(+)CD25(+)Foxp3(+) regulatory cells (Tregs) are a special lineage of cells central in the maintenance of immune homeostasis, and are targeted for human immunotherapy. They are conventionally associated with the production of classical anti-inflammatory cytokines such as IL-10, TGF-ß and IL-35, consistent to their anti-inflammatory functions. However, emerging evidence show that they also express effector cytokines such as IFN-γ and IL-17A under inflammatory conditions. While some studies reveal that these pro-inflammatory cytokine producing Foxp3(+) regulatory cells retain their suppressive ability, others believe that these cells are dys-regulated and are associated with perpetuation of immunopathology. Therefore the development of these cells may challenge the efficacy of human Treg therapy. Mechanistically, toll-like receptor (TLR) ligands and the pro-inflammatory cytokine milieu have been shown to play important roles in the induction of effector cytokines in Tregs. Here we review the mechanisms of development and the possible functions of pro-inflammatory cytokine producing Foxp3+ Tregs.

Th17 inflammation model of oropharyngeal candidiasis in immunodeficient mice.

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro cultured Th17 cells, followed by oral infection with Candida albicans (C. albicans). Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (Tregs) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.

TLR-2 Signaling Promotes IL-17A Production in CD4+CD25+Foxp3+ Regulatory Cells during Oropharyngeal Candidiasis.

Recent studies show that CD4+CD25+Foxp3+ regulatory cells (Tregs) produce effector cytokines under inflammatory conditions. However, the direct role of microbial agents that serve as toll-like receptor (TLR) ligands in the induction of effector cytokines in Tregs is less clear. Here we show that CD4+Foxp3+Tregs produce the effector cytokine IL-17A during oropharyngeal candidiasis (OPC) and inflammatory bowel disease in a TLR-2/Myd88 signaling dependent manner. TLR-2 ligands promote proliferation in Tregs in the presence and absence of TCR signals and inflammatory cytokines in vitro. The proliferation is directly dependent on TLR-2 expression in Tregs. Consistent with this, Tlr2-/- mice harbor fewer thymically derived Tregs and peripheral Tregs under homeostatic conditions in vivo. However, under Th17 inducing conditions, IL-6 and TLR-2 signaling both in Tregs as well as antigen presenting cells (APC) are critical for maximal ROR-γt and IL-17A up-regulation in Foxp3+ Tregs. The minimal and transient loss of Foxp3 expression and suppressive properties are due to the presence of IL-6 in the milieu, but not the direct effect of TLR-2 signaling in Tregs. Taken together, our data reveal that TLR-2 signaling promotes not only proliferation, but also IL-17A in Tregs, depending on the cytokine milieu. These IL-17A producing Tregs may be relevant in mucosal infections and inflammation.